1. Field of the Invention
This invention relates to the production of bioadhesives that can be employed to bond substances for use in wet environments. Typically, the bioadhesives of the invention are employed as marine adhesives, biomedical adhesives or dental adhesives. The invention further relates to the microbial production of bioadhesive precursor proteins that can be converted to bioadhesives by chemical or enzymatic treatment.
2. Brief Description of Background Art
The properties of adhesives generally must be tailored to meet the requirements of the particular environments in which they are to be used. Ideally, an adhesive should be cured and it should maintain both its adhesivity and cohesivity under the conditions of use. Curing is the altering of the physical properties of an adhesive by chemical or enzymatic means. In the case of the bioadhesives produced by the procedures described herein, curing is likely to be due to the cross-linking of adjacent uncured adhesive molecules by catalytic and/or chemical agents. Curing may also involve adhesive cross-linking with the substrate.
Many adhesives that exhibit excellent adhesive properties under dry conditions suffer a substantial or total loss of those properties in wet environments. Furthermore, adhesives of the prior art cannot be cured in wet environments. Consequently, it has been particularly difficult to develop adhesives for use in wet environments, such as marine adhesives or adhesives for use in medical and dental applications.
Marine mussels and other sessile invertebrates have the ability to secrete adhesive substances by which they affix themselves to underwater objects. For example, mussels of the genus Mytilus, e.g., the species Mytilus edulis and Mytilus californianus, deposit an adhesive substance from the mussel foot that becomes cured, forming a permanent attachment to the substrate. A major component of the adhesive plaque deposited by M. edulis has been identified as a hydroxylated protein of about 130,000 daltons (Waite, J. H., J. Biol. Chem., 258:2911-2915 (1983)). While this substance might make an excellent adhesive for use in wet environments, isolation of the uncured adhesive from mussels for commercial use is not practical, since the extraction of 1 kg of the adhesive substance would require the use of about 5 to 10 million mussels. Besides involving a very laborious process, great care would have to be taken, in case the adhesive were to be employed in medical applications, to insure that other mussel proteins and contaminants were removed from the bioadhesive prior to use in order to prevent antigenic or anaphylactic reactions.
U.S. Pat. No. 4,585,585 describes a procedure for preparing a bioadhesive polymer by chemically linking decapeptide units produced by the enzymatic digestion of isolated mussel adhesive protein. In accordance with the disclosure of that patent, a bioadhesive protein is first isolated from phenol glands of mussels of the genus Mytilus using the protein purification procedures described by Waite and Tanzer in Science, 212:1038 (1981). The isolated bioadhesive, having a molecular weight of 120,000 to 140,000 daltons, is first treated with collagenase, which reduces its molecular weight by about 10,000 daltons. The treated protein is then digested with trypsin, and the digested protein subjected to gel filtration dialysis to isolate decapeptides of the general formula EQU NH.sub.2 -Ala-Lys-Pro/Hyp-Ser/Thr-Tyr/Dopa-Pro/Hyp-Pro/Hyp-Ser/Thr-Tyr/Dopa-Lys-COO H
The decapeptides produced in this manner are then polymerized by the use of chemical linking groups such as glutaraldehyde, oligopeptides, amino acids or other bifunctional linking groups to produce bioadhesives containing up to about 1,000 such decapeptide units.
The procedure of U.S. Pat. No. 4,585,585 still requires the isolation of bioadhesive protein from mussel, which, as previously indicated, is impractical on a commercial scale. Moreover, in addition to the laborious purification procedure, this process adds the additional steps of enzymatic digestion, isolation of the decapeptide fragments and chemical reassemblage of the fragments into a bioadhesive polymer. This arduous procedure is not well-suited to commercial production. Further, the polymers produced by this method are quite different from the mussel adhesive since they contain only chemically polymerized decapeptides produced by trypsin digestion of the natural molecule. Analysis of the natural gene described herein demonstrates that there are other significant sequence elements in the mussel adhesive from Mytilus edulis.
Thus a need has continued to exist for means and methods for the efficient production of bioadhesives having the excellent properties associated with the mussel foot bioadhesive in wet environments.
A further need has continued to exist for means and methods for producing bioadhesives having the properties of the mussel foot adhesive without the necessity of handling and processing large quantities of mussels.